Anti Cancer Pre Screening for Several Pl
Anti Cancer Pre-Screening for Several Plant Using
Brine Shrimp Lethality Test
Chasanah U1., H. Rachmawati1, V Febriana1, F.T. Wulandari1, S.B. Sikdewa1, R.
Amalia1, A.S. Debby1, A.S. Jamil1
Program Studi Farmasi Fakultas Ilmu Kesehatan Universitas Muhammadiyah Malang
email contact: [email protected]
Abstract. Indonesia has high biodiversity especially its pharmacologically beneficial flora such as
flora with anti cancer activity. The aim of this study is to screen several plant candidate for anti
cancer therapy. Brine shrimp lethality test (BST) is the method that used in this study. By measuring
the LC50 of the several plant extract to the Artemia salina larvae that analogue with the cancer cells.
The result from the fiftht plant were tested show various responses. With n-Hexane extract of Physalis
minima, Allium odoratum, Tabebuia chrysantha has potential biological (anticancancer) activity with
LC50 920,92 µg/ml, 805,62 µg/ml, and 652.868 µg/ml. And with methanol extract Chentotecha
longilamina, Impatiens balsamina, Tabebuia chrysantha.shows the activity each with LC50 908,58 µg
/ ml, 744,48 µg/ml and 858 µg/ml.
Key Word: anticancer activity, BST, plants
1. Background
Cancer is still became most dangerous disease in the world. Based on World Health
Organization in 2005; 7,6 million people death by cancer and 84 million people is going
to be death 10 years onward (Diananda, 2007). At the advance country the cancer is the
second disease that causing death after the cardiovascular diseases (Nafrialdi and
Sulistia, 2007). As a development country the cancer diseases in indonesia being the
sixth disease with high mortality number.
In the other word, indonesia has many potential in its natural resources, especially its
floral biodiversity that has many potential as natural drugs. Mangan (200), from 30.000
.floral species in Indonesia, about 1.260 species has pharmacological activities including
anti cancer. Based on the potency of the floral species in Indonesia, the aim of this
research are to get several potential plant for anticancer therapy.
The plant candidates were chosen by study its chemotaxonomy with plants that has close
relationship in its chemical compound and its morphological taxonomy. Physalis
minima, Allium odoratum, Tabebuia chrysantha, Chentotecha longilamina, Impatiens
balsamina are plants that chosen to know its anticancer activity. Its chosen by literally
study its morphological and chemical compound relation with the former plant that has
know has anti cancer activity. Our effort to identify the bioactive compound of the
plants we use bioassay test. Brine Shrimph Brine shrimp lethality test (BST) has been
employed as an alternative bioassay technique to screen the plant extracts (Meyer et al.,
1982; McLaughlin and Rogers, 1998).
1
2
2. Materials and Methods
2.1. Plants Material
All plant materials were collected from several area Malang East Java Indonesia.
Physalis minima (all part), Allium odoratum (all part), Tabebuia chrysantha (caulis
cortex), Chentotecha longilamina (all part), Impatiens balsamina (all part). Plant
determination conducted in Balai Materia Medica Batu City and Purwodadi National
Park.
2.2. Preparation of Plants Extract
Five hundred grams (500 g) of the plant powder were macerated with two solvent
each 1,5 liters. First solvent n-hexane, the filtrate and residue from n-hexane
maceration separated. The dry residues are macerate once again with methanol and
separated to get the filtrate. Both filtrates evaporated with rotary evaporator with 40oC
then dried with oven 40oC until gained dry extract.
2.3. Brine Shrimp Lethality Test
Brine shrimp lethality bioassay was carried out to investigate the cytoxicity of plant
extracts. Artemia salina (Leach) eggs were added to a hatching chamber containing
Ocean/Sea water. The hatching chamber was kept under an inflorescent bulb for 48h
for the eggs to hatch into shrimp larvae. Different concentration of plant extract
prepared by diluting extract with solvent 10, 50, 100, 500, and 100 µg/ml for both nhexane extract and methanol extract. Four and a half millilitre (4,5ml) of Ocean/Sea
water was added to each vial, and 10 larvae of A. salina Leach (taken 48 – 72h after
the initiation of hatching) were added to each vial. The final volume of solution in
each vial was adjusted to 5 ml with Ocean/Sea water immediately after adding the
shrimps. One drop of dimethyl sulphoxide (DMSO) was added to the test and control
vials before adding the shrimps to enhance the solubility of test materials. Each
treatment is triplicate. LC50 values were determined at 95% confidence intervals by
analyzing the data with statistical analysis. The LC50 values of the brine shrimps
obtained for extracts of the plants studied were recorded.
2.4. Phytochemical Screening
The Phyto-chemist screening of bioactive compound of the plants including
flavonoid, saponin Glycoside, Steroid Triterpenoid, Alkaloid, Tannin and Poliphenol
screening.
3. Result and Discussion
The result from the fifth plant were tested show various responses. With n-Hexane
extract of Physalis minima, Allium odoratum, Tabebuia chrysantha has potential
biological (anticancancer) activity with LC50 920,92 µg/ml, 805,62 µg/ml, and 652,868
µg/ml. And with methanol extract Chentotecha longilamina, Impatiens balsamina,
Tabebuia chrysantha shows the activity each with LC50 908,58 µg / ml, 744,48 µg/ml
and 858 µg/ml (Table 1).
According to Meyer et al. (1982) several naturally extracted products which had LC50 <
1000 µg/mL using brine shrimp bioassay were known to contain physiologically active
principles. Although toxicity test using BST does not give a clear depiction on
cytotoxicity against cancer cell, however this method has been reported useful for
screening anticancer from plant/natural sources.
3
The LC50 result from Physalis minima, Allium odoratum, with n-hexane extract shows
that the more potential bioactive compound from this plants are soluble in non polar
solvent than polar solvent. But from Chentotecha longilamina, Impatiens balsamina are
vice versa. One plant under this research that has potential both in polar and non polar
solvent are Tabebuia chrysantha 858 µg/ml
Table 1. Brine Shrimp Test of Cytotoxicity of plant extract under study
n-Hexane
LC50
Methanol
LC50
all part
920,92
1281,39
Allium odoratum
all part
805,62
1005,75
Tabebuia chrysantha
cortex of stem
652,87
858,63
Chentotheca longilamina
all part
2193,26
908,58
Impatiens balsamina
all part
1167,75
744,49
Plant
Part Used
Physalis minima
Table 2. Result from phytochemical screening from plant bioactive substances
Screening Result
Plant
Physalis minima
Flavonoid
n
Methanol
Hexan
+
X
+
X
Tabebuia chrysantha
+
Chentotheca longilamina
X
Impatiens balsamina
X
Allium odoratum
Glycoside saponin
n
Methanol
Hexan
+
X
+
X
X
-
+
X
+
X
Steroid Triterpenoid
n
Methanol
Hexan
+
X
+
X
X
+
-
X
-
X
Tannin & Poliphenol
n
Methanol
Hexan
X
-
X
X
+
X
+
X
-
+
X
+
*X: not tested
According to Table 2, there are shows that for Physalis minima and Allium odoratum nHexane extract. Its bioactive compound which has cytotoxicity effect has flavonoid, saponin
glycoside, and steroid triterpen content. Chentotheca longilamina and Impatiens balsamina
both has Saponin Glycoside and Steroid triterpen content but plus Tannin & polyphenol in I.
balsamina. Also in Tabebuia chrysantha look has flavonoid, steroid triterpenoid and tannin &
polyphenol.
All plant above has steroid/triterpenoid substances (Table 2). This maybe the main factor for
that plants has cytotoxic activity and potential as anti cancer agents. Thoppil and Bhisayee
(2011) mention that terpenoid has potential as chemopreventive and therapeutic for cancer.
Former identified terpenoid such as Geraniol and Limonene are stated has anti cancer
activity. Geraniol, an acyclic dietary monoterpene, represents the only monoterpene that has
been studied in vitro against liver cancer cells. Geraniol was shown to inhibit the growth of
HepG2 human hepatic carcinoma cells by decreasing 3-hydroxymethylglutaryl coenzyme A
(HMG-CoA) reductase, the major rate-limiting enzyme in cholesterol biosynthesis in
mammals (Thoppil and Bhisayee, 2011).
From Table 2. there are also shows that all plants extract has flavonoid content. Ren et al.
(2003) mentioned that many mechanisms of action have been identified from flavonoid,
including carcinogen inactivation, antiproliferation, cell cycle arrest, induction of apoptosis
and differentiation, inhibition of angiogenesis, antioxidation and reversal of multidrug
4
resistance or a combination of these mechanisms. Based on these results, flavonoids may be
promising anticancer agents.
4. Conclusion
Based on this reseach, it can be conclude that all the plants (Physalis minima, Allium
odoratum, Tabebuia chrysantha, Chentotecha longilamina, Impatiens balsamina) has
cytotoxic activity and potential to become anti cancer agent. From these plant also know
triterpenoid and flavonoid can be the main substance which has the cytotoxic activity and
potential as anticancer agent. Based on this research there are good chanche for researchers to
continue this topic deeper. Analyzing and puryfing the bioactive compound and testing it to
human cell line in vitro and propably testing it invivo and step forward with clinical trial if
necesarry.
5. Acknowledgement
We thank to Prof. Sukardiman, Apt, MS from Department of Natural Resources, Faculty of
Pharmacy, Airlangga University for the guidance and providing several needs for this
research.
6. Reference
1. Dalimartha, Setiawan, 2004. Ramuan Tradisional Untuk Pengobatan Kanker,
Penebar Swadaya, Jakarta.
2. Mangan, Yellia., 2009. Solusi Sehat Mencegah dan Mengatasi Kanker. Cetakan ke-1,
Jakarta : Penerbit Agromedia Pustaka, hal 1-36.
3. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsey, L.B., Nichols, D.E. and
McLaughlin, J.L. (1982). Brine Shrimp: A convenient general bioassay for active plant
constituents. Planta Medica 45:31 – 34.
4. Mclaughlin, J. L., And L. L. Rogers. 1998, The Use Of Biological Assays To Evaluate
Botanicals Drug Information Journal, Vol. 32, pp. 513–524,Nafrialdi dan Sulistia,G.,
Antikanker, Farmakologi dan Terapi, Edisi 5, Jakarta: Bagian Farmakologi Fakultas
Kedokteran Universitas Indonesia, hal. 732-733.
5. Roslin J Thoppil, Anupam Bishayee 2011. Terpenoids as potential chemopreventive
and therapeutic agents in liver cancer World J Hepatol 2011 September 27; 3(9): 2286. Ren, W., Zhenhua Qiao, Hongwei Wang, Lei Zhu, Li Zhang 2003. Flavonoids:
Promising Anticancer Agents Medicinal Research Reviews, Vol. 23, No. 4, 519-534.
Brine Shrimp Lethality Test
Chasanah U1., H. Rachmawati1, V Febriana1, F.T. Wulandari1, S.B. Sikdewa1, R.
Amalia1, A.S. Debby1, A.S. Jamil1
Program Studi Farmasi Fakultas Ilmu Kesehatan Universitas Muhammadiyah Malang
email contact: [email protected]
Abstract. Indonesia has high biodiversity especially its pharmacologically beneficial flora such as
flora with anti cancer activity. The aim of this study is to screen several plant candidate for anti
cancer therapy. Brine shrimp lethality test (BST) is the method that used in this study. By measuring
the LC50 of the several plant extract to the Artemia salina larvae that analogue with the cancer cells.
The result from the fiftht plant were tested show various responses. With n-Hexane extract of Physalis
minima, Allium odoratum, Tabebuia chrysantha has potential biological (anticancancer) activity with
LC50 920,92 µg/ml, 805,62 µg/ml, and 652.868 µg/ml. And with methanol extract Chentotecha
longilamina, Impatiens balsamina, Tabebuia chrysantha.shows the activity each with LC50 908,58 µg
/ ml, 744,48 µg/ml and 858 µg/ml.
Key Word: anticancer activity, BST, plants
1. Background
Cancer is still became most dangerous disease in the world. Based on World Health
Organization in 2005; 7,6 million people death by cancer and 84 million people is going
to be death 10 years onward (Diananda, 2007). At the advance country the cancer is the
second disease that causing death after the cardiovascular diseases (Nafrialdi and
Sulistia, 2007). As a development country the cancer diseases in indonesia being the
sixth disease with high mortality number.
In the other word, indonesia has many potential in its natural resources, especially its
floral biodiversity that has many potential as natural drugs. Mangan (200), from 30.000
.floral species in Indonesia, about 1.260 species has pharmacological activities including
anti cancer. Based on the potency of the floral species in Indonesia, the aim of this
research are to get several potential plant for anticancer therapy.
The plant candidates were chosen by study its chemotaxonomy with plants that has close
relationship in its chemical compound and its morphological taxonomy. Physalis
minima, Allium odoratum, Tabebuia chrysantha, Chentotecha longilamina, Impatiens
balsamina are plants that chosen to know its anticancer activity. Its chosen by literally
study its morphological and chemical compound relation with the former plant that has
know has anti cancer activity. Our effort to identify the bioactive compound of the
plants we use bioassay test. Brine Shrimph Brine shrimp lethality test (BST) has been
employed as an alternative bioassay technique to screen the plant extracts (Meyer et al.,
1982; McLaughlin and Rogers, 1998).
1
2
2. Materials and Methods
2.1. Plants Material
All plant materials were collected from several area Malang East Java Indonesia.
Physalis minima (all part), Allium odoratum (all part), Tabebuia chrysantha (caulis
cortex), Chentotecha longilamina (all part), Impatiens balsamina (all part). Plant
determination conducted in Balai Materia Medica Batu City and Purwodadi National
Park.
2.2. Preparation of Plants Extract
Five hundred grams (500 g) of the plant powder were macerated with two solvent
each 1,5 liters. First solvent n-hexane, the filtrate and residue from n-hexane
maceration separated. The dry residues are macerate once again with methanol and
separated to get the filtrate. Both filtrates evaporated with rotary evaporator with 40oC
then dried with oven 40oC until gained dry extract.
2.3. Brine Shrimp Lethality Test
Brine shrimp lethality bioassay was carried out to investigate the cytoxicity of plant
extracts. Artemia salina (Leach) eggs were added to a hatching chamber containing
Ocean/Sea water. The hatching chamber was kept under an inflorescent bulb for 48h
for the eggs to hatch into shrimp larvae. Different concentration of plant extract
prepared by diluting extract with solvent 10, 50, 100, 500, and 100 µg/ml for both nhexane extract and methanol extract. Four and a half millilitre (4,5ml) of Ocean/Sea
water was added to each vial, and 10 larvae of A. salina Leach (taken 48 – 72h after
the initiation of hatching) were added to each vial. The final volume of solution in
each vial was adjusted to 5 ml with Ocean/Sea water immediately after adding the
shrimps. One drop of dimethyl sulphoxide (DMSO) was added to the test and control
vials before adding the shrimps to enhance the solubility of test materials. Each
treatment is triplicate. LC50 values were determined at 95% confidence intervals by
analyzing the data with statistical analysis. The LC50 values of the brine shrimps
obtained for extracts of the plants studied were recorded.
2.4. Phytochemical Screening
The Phyto-chemist screening of bioactive compound of the plants including
flavonoid, saponin Glycoside, Steroid Triterpenoid, Alkaloid, Tannin and Poliphenol
screening.
3. Result and Discussion
The result from the fifth plant were tested show various responses. With n-Hexane
extract of Physalis minima, Allium odoratum, Tabebuia chrysantha has potential
biological (anticancancer) activity with LC50 920,92 µg/ml, 805,62 µg/ml, and 652,868
µg/ml. And with methanol extract Chentotecha longilamina, Impatiens balsamina,
Tabebuia chrysantha shows the activity each with LC50 908,58 µg / ml, 744,48 µg/ml
and 858 µg/ml (Table 1).
According to Meyer et al. (1982) several naturally extracted products which had LC50 <
1000 µg/mL using brine shrimp bioassay were known to contain physiologically active
principles. Although toxicity test using BST does not give a clear depiction on
cytotoxicity against cancer cell, however this method has been reported useful for
screening anticancer from plant/natural sources.
3
The LC50 result from Physalis minima, Allium odoratum, with n-hexane extract shows
that the more potential bioactive compound from this plants are soluble in non polar
solvent than polar solvent. But from Chentotecha longilamina, Impatiens balsamina are
vice versa. One plant under this research that has potential both in polar and non polar
solvent are Tabebuia chrysantha 858 µg/ml
Table 1. Brine Shrimp Test of Cytotoxicity of plant extract under study
n-Hexane
LC50
Methanol
LC50
all part
920,92
1281,39
Allium odoratum
all part
805,62
1005,75
Tabebuia chrysantha
cortex of stem
652,87
858,63
Chentotheca longilamina
all part
2193,26
908,58
Impatiens balsamina
all part
1167,75
744,49
Plant
Part Used
Physalis minima
Table 2. Result from phytochemical screening from plant bioactive substances
Screening Result
Plant
Physalis minima
Flavonoid
n
Methanol
Hexan
+
X
+
X
Tabebuia chrysantha
+
Chentotheca longilamina
X
Impatiens balsamina
X
Allium odoratum
Glycoside saponin
n
Methanol
Hexan
+
X
+
X
X
-
+
X
+
X
Steroid Triterpenoid
n
Methanol
Hexan
+
X
+
X
X
+
-
X
-
X
Tannin & Poliphenol
n
Methanol
Hexan
X
-
X
X
+
X
+
X
-
+
X
+
*X: not tested
According to Table 2, there are shows that for Physalis minima and Allium odoratum nHexane extract. Its bioactive compound which has cytotoxicity effect has flavonoid, saponin
glycoside, and steroid triterpen content. Chentotheca longilamina and Impatiens balsamina
both has Saponin Glycoside and Steroid triterpen content but plus Tannin & polyphenol in I.
balsamina. Also in Tabebuia chrysantha look has flavonoid, steroid triterpenoid and tannin &
polyphenol.
All plant above has steroid/triterpenoid substances (Table 2). This maybe the main factor for
that plants has cytotoxic activity and potential as anti cancer agents. Thoppil and Bhisayee
(2011) mention that terpenoid has potential as chemopreventive and therapeutic for cancer.
Former identified terpenoid such as Geraniol and Limonene are stated has anti cancer
activity. Geraniol, an acyclic dietary monoterpene, represents the only monoterpene that has
been studied in vitro against liver cancer cells. Geraniol was shown to inhibit the growth of
HepG2 human hepatic carcinoma cells by decreasing 3-hydroxymethylglutaryl coenzyme A
(HMG-CoA) reductase, the major rate-limiting enzyme in cholesterol biosynthesis in
mammals (Thoppil and Bhisayee, 2011).
From Table 2. there are also shows that all plants extract has flavonoid content. Ren et al.
(2003) mentioned that many mechanisms of action have been identified from flavonoid,
including carcinogen inactivation, antiproliferation, cell cycle arrest, induction of apoptosis
and differentiation, inhibition of angiogenesis, antioxidation and reversal of multidrug
4
resistance or a combination of these mechanisms. Based on these results, flavonoids may be
promising anticancer agents.
4. Conclusion
Based on this reseach, it can be conclude that all the plants (Physalis minima, Allium
odoratum, Tabebuia chrysantha, Chentotecha longilamina, Impatiens balsamina) has
cytotoxic activity and potential to become anti cancer agent. From these plant also know
triterpenoid and flavonoid can be the main substance which has the cytotoxic activity and
potential as anticancer agent. Based on this research there are good chanche for researchers to
continue this topic deeper. Analyzing and puryfing the bioactive compound and testing it to
human cell line in vitro and propably testing it invivo and step forward with clinical trial if
necesarry.
5. Acknowledgement
We thank to Prof. Sukardiman, Apt, MS from Department of Natural Resources, Faculty of
Pharmacy, Airlangga University for the guidance and providing several needs for this
research.
6. Reference
1. Dalimartha, Setiawan, 2004. Ramuan Tradisional Untuk Pengobatan Kanker,
Penebar Swadaya, Jakarta.
2. Mangan, Yellia., 2009. Solusi Sehat Mencegah dan Mengatasi Kanker. Cetakan ke-1,
Jakarta : Penerbit Agromedia Pustaka, hal 1-36.
3. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsey, L.B., Nichols, D.E. and
McLaughlin, J.L. (1982). Brine Shrimp: A convenient general bioassay for active plant
constituents. Planta Medica 45:31 – 34.
4. Mclaughlin, J. L., And L. L. Rogers. 1998, The Use Of Biological Assays To Evaluate
Botanicals Drug Information Journal, Vol. 32, pp. 513–524,Nafrialdi dan Sulistia,G.,
Antikanker, Farmakologi dan Terapi, Edisi 5, Jakarta: Bagian Farmakologi Fakultas
Kedokteran Universitas Indonesia, hal. 732-733.
5. Roslin J Thoppil, Anupam Bishayee 2011. Terpenoids as potential chemopreventive
and therapeutic agents in liver cancer World J Hepatol 2011 September 27; 3(9): 2286. Ren, W., Zhenhua Qiao, Hongwei Wang, Lei Zhu, Li Zhang 2003. Flavonoids:
Promising Anticancer Agents Medicinal Research Reviews, Vol. 23, No. 4, 519-534.